During ensiling process, microbial populations present in forage mass consume forage carbohydrates as their energy source and produce different metabolite such as lactic acid, acetic acid and butyric acid. Lactic acid production by Lactobacillus species decreases the pH of silage (Kung and Ranjit, 2001) which could restrict activity of undesirable microorganism such as Clostridia (Duniere et al. 2011). Clostridia convert forage carbohydrates to butyric acid which could lead to increase in pH and further spoilage of the silage (Dunière et al. 2013). In order to ensure Lactobacillus predominating fermentation and high concentration of lactic acid in silage, various additives were tested (Dunie`re et al. 2013; Yitbarek and Tamir, 2014). Essential oils are plant secondary metabolites (PSM) which are responsible for the odor and color of them (Balandrin and Klocke, 1985) and commonly extracted from different plant tissues by distillation methods (Losa, 2001). Many studies reported the stimulatory and inhibitory effects of essential oils on vast variety of microorganisms under in vitro and in vivo conditions (Newbold et al. 2004; Benchaar et al. 2007; Nanon et al. 2014; Khorrami et al. 2015). For example, Fraser et al. (2007) evaluated the effects of cinnamon essential oil on rumen microbial fermentation using continuous culture system and observed a reduction in ammonia nitrogen (NH3-N) concentration of cultural medium. McIntosh et al. (2003) also observed inhibition of hyper ammonia-producing bacteria by a commercial blend of essential oils. Moreover, some of former studies demonstrated the potential of these compounds to alter rumen fermentation by reducing the proportion of acetate to propionate and also inhibition of methanogenesis (Busquet et al. 2005; Benchaar and Greathead, 2011). All of these studies used essential oils directly in ruminant feed or in cultural medium. Based on available information, very limited numbers of studies have assessed the effects of some essential oils on silage fermentation. Kung et al. (2008) supplemented corn silage with different doses of a specific blend of essential oils and observed no improvement in silage fermentation characteristics and in aerobic stability of corn silage. However, in study of Chaves et al. (2012), addition of cinnamon leaf, oregano and sweet orange essential oils in barley silage decreased yeast growth during aerobic stability test. The present study was conducted to determine the effects of essential oils of cinnamon, thyme, mint, oregano or cumin on chemical composition, aerobic stability and fermentation characteristics of corn silage and to evaluate the effect of the silages on in vitro rumen fermentation pattern and methane production using gas production technique.
MATERIALS AND METHODS
Essential oils preparation
Cinnamon (C. verum), thyme (T. vulgaris), mint (M. spicata), oregano (O. vulgare) and cumin (C. cyminum) used in this study were purchased from local markets. The plant obtained was cut or crushed into small pieces, oven-dried at 39 ˚C for 48 h and ground to pass a 1 mm-screen. Essential oils content of each plant was obtained with hydro-distillation of grinded samples using Clevenger apparatus (Jahani-Azizabadi et al. 2014). The essential oils were stored in refrigerator (4 ˚C) until they were used in the experiment.
Whole crop corn was sampled every other day to measure dry matter (DM) content by air forced oven (65 ˚C for 48 h) and harvested when reached at 29%. Then it was chopped at 20 mm length and ensiled in laboratory scale polyvinyl tubes (4±0.25 kg) for 45 days. The forage treated with no additives (control) or treated with essential oils at the rate of 120 or 240 mg kg-1 DM. Essential oils used were cinnamon (Cinn120 or Cinn240, respectively), thyme (Thy120 or Thy240, respectively), mint (Mint120 or Mint240, respectively), oregano (Oreg120 or Oreg240, respectively) and cumin (Cum120 or Cum240, respectively). Each treatment was replicated in a quadruplicate manner. All additives were dissolved in 0.5 v/v aqueous ethanol (Chaves et al. 2012) and sprayed onto the chopped forages. The same amount of the ethanol was also added to the control.
After silo opening, 2 kg samples of each replicate from each treatment were placed loosely into a clean, 20 L bucket. Silages were exposed to air at room temperature and thermometers were placed in the center of the silage masses. A double layer of cheesecloth was placed over each container to prevent drying and contamination, but allowing penetration of air. Ambient temperature and the temperature from each silage were recorded every 2 h. Temperatures were monitored for 12 days. Aerobic stability was defined as the number of hours the silage remained stable before a 2 ˚C increase in temperature above the ambient temperature (Moran et al. 1996).
Gas production technique
One liter of rumen liquor was obtained before the morning feeding from two ruminally fistulated lactating Holstein dairy cows. Animals were fed (g/kg, DM basis): alfalfa hay, 240; corn silage, 160; steam flaked corn grain, 210; whole linted cottonseed, 120 and a barley grain-based concentrate, 270 [19% crude protein (CP), 34% neutral detergent fiber (NDF) and 1.6 Mcal/kg NEl]. Pooled rumen fluid was squeezed through 4 layers of cheesecloth into an insulated thermos. Under continuous flushing of CO2, 30 mL of buffered rumen fluid (ratio of buffer to rumen fluid was 2:1, buffer were prepared as proposed by Menke and Steingass, 1988) was dispensed with pump into a 125 mL serum bottle containing 250 mg dried of each experimental silage. Eight bottles were considered for each treatment in two separate runs. Bottles were sealed with rubber stopper and aluminum cap and placed in a shaking water bath for 24 h at 38.6 ˚C. At the end of incubation, gas pressure was measured by a pressure transducer and converted into volume using an experimentally calibrated curve (Theodorou et al. 1994). Methane production was determined using a multiple gas detector (SR2-BIO System, SEWERIN, Germany). Finally, the content of each bottle were filtered and the residue was oven dried (65 ˚C for 48 h) and used to calculate in vitro dry matter disappearance (IVDMD). Partitioning factor (PF) was calculated as mg DM disappeared per ml accumulative gas produced within 24 hours post incubation.
Dry matter content of the silages was determined by air forced oven (65 ˚C for 48 h). Dried silage samples were ground to pass through a 1 mm-screen for later analysis. Crude protein was measured according to the Kjeldahl procedure (AOAC, 1990) on the Tecator Auto-analyzer (Kjeltec 2300 Auto-analyzer, Foss Tecator AB, Hoganas, Sweden). Determination of NDF was done according to Van Soest et al. (1991). Aqueous extract were prepared from ensiled samples by mixing 50 g of forage with 450 mL of deionized water and homogenizing this mix for 1 min. Then, silage pH was determined using a portable pH meter. A portion of aqueous extracts were filtered through four layers of cheesecloth and acidified with 0.2 N HCl (1:1). NH3-N concentration of acidified silage extracts were determined using distillation method.
The effects of type of essential oils regardless of applied level (mathematical mean of both levels) and also different levels of each essential oil compared with the control were tested separately using general linear models (GLM) procedure of SAS (2002). The model used for each of the analysis was as follows:
Yij= μ + Ti + eij
Yij: dependent variable.
μ: population mean for the variable.
Ti: effect of treatment i.
eij: random error associated with the observation ij.
When the overall F-test was significant, differences between means and the control were declared significant at (P<0.05) using the Dunnett comparison test.
RESULTS AND DISCUSSION
Chemical composition, pH, NH3-N concentration (mg dL-1) and aerobic stability of the experimental silages based on the type of applied essential oils are shown in Figure 1 and Table 1. Higher (P<0.05) DM content was observed in Cinn120 or Cinn240 compared with the control (Table 1). Relative to the control, CP concentration of silages supplemented with mint or oregano essential oils increased significantly (P<0.05). Application of thyme essential oil significantly suppressed NDF of silage compared with the control (P<0.05). Cinn240 and Cum240 also had lower NDF concentration compared with the control (P<0.05). Furthermore, significant increment in NDF concentration of Mint120 and Oreg240 was observed compared with the control (P<0.05). The pH value of Mint120 and Mint240 was significantly higher and lower than the control, respectively (P<0.05). Moreover, addition of oregano or cumin essential oils to the corn silage increased its pH value relative to the control (P<0.05). A significant reduction in NH3-N concentration of silages treated with thyme essential oil was observed in comparison to the control. On the other hand, NH3-N concentration of Mint120, Cum240, Oreg120 and Oreg240 were arisen compared with the control (P<0.05). All of applied essential oils except of mint caused a significant improvement in silage aerobic stability compared with the untreated silage (ranging from 44% to 400% increment). Total gas and methane production, IVDMD and PF of the experimental silages based on the type of applied essential oils are shown in Figure 2. In addition, all of these parameters are shown in Table 2 based on the different levels of each essential oil. Relative to the control, only total produced gas from Oreg240 was diminished (P<0.05) and Cinn240, Thy120, Thy240 or Cum120 produced more gas (P<0.05) 24 h post incubation (Table 2). When compared with the control, all treated silages except of Mint240 or Oreg120 produce higher methane after 24 h incubation (P<0.05). However, the ratio of methane to total gas was not affected by experimental treatments (P>0.05). Compared with the control, IVDMD of Thy120, Mint240 or Oreg240 increased by 10 percent (P<0.05). On the contrary, Cinn120, Oreg120, Cum120 or Cum240 had lower IVDMD compared with the control. When compared with the control, an improvement was observed in PF of Oreg240, whereas it was reduced in silages treated with cinnamon or cumin essential oils (P<0.05). Silages treated with cinnamon essential oil were characterized by higher DM content. This phenomenon could be due to the limitation of development of special groups of microorganism and therefore smaller loss of nutrients (Selwet, 2009). In the current study, silages treated with mint or oregano essential oils had higher CP concentration. Protein degradation in silage is consequent of proteolytic microorganisms, such as clostridia or enterobacteria present on the plant (McDonald et al. 1991). Inhibitory effects of some essential oils including oregano on growth of clostridia were reported in previous studies (Ismaiel and Pierson, 1990).
Figure 1 Chemical composition, fermentation characteristics and aerobic stability of corn silages treated with different essential oils (without consideration of applied levels). [(a) dry matter, (b) crude protein, (c) neutral detergent fiber, (d) pH, (e) ammonia nitrogen and (f) aerobic stability]
* Bars with asterisk differ significantly from the control (P<0.05)
On the other hand, higher CP content of silages containing oregano essential oils did not confirm findings of Chaves et al. (2012) who observed no change in CP of barley silage supplemented with oregano. This is because of differences in the applied concentrations and possibly the type of silages (Hart et al. 2008). Higher pH value in most of the treated silage could be the result of reduction in activity of Lactobacillus bacteria, as lactic acid production by these species could decline the pH of the silage (Kung and Ranjit, 2001). However, in two previous studies, essential oils had no inhibitory effect on lactic acid producing bacteria (Kung et al. 2008; Chaves et al. 2012).
Table 1 The effects of different essential oil on overall composition of corn silage
Control: corn silage with no additive; Cinn120: corn silage treated with 120 mg cinnamon essential oil per kg dry matter; Cinn240: corn silage treated with 240 mg cinnamon essential oil per kg dry matter; Thy120: corn silage treated with 120 mg thyme essential oil per kg dry matter; Thy240: corn silage treated with 240 mg thyme essential oil per kgdry matter; Mint120: corn silage treated with 120 mg mint essential oil per kg dry matter; Mint240: corn silage treated with 240 mg mint essential oil per kg dry matter; Oreg120: corn silage treated with 120 mg oregano essential oil per kgdry matter; Oreg240: corn silage treated with 240 mg oregano essential oil per kg dry matter; Cum120: corn silage treated with 120 mg cumin essential oil per kg dry matter and Cum240: corn silage treated with 240 mg cumin essential oil per kg dry matter.
DM: dry matter; CP: crude protein and NDF: neutral detergent fiber.
SEM: standard error of the means.
Figure 2 In vitro ruminal gas production characteristics of corn silages treated with different essential oils (without consideration of applied levels) after 24h incubation. [(a) total gas production, (b and c) methane production, (d) in vitro dry matter disappearance and (e) partitioning factor]
1 mg DM disappeared/mL gas produced
* Bars with an asterisk differ significantly from the control (P<0.05)
Table 2 In vitro ruminal fermentation of corn silages treated with different essential oils after 24 h of incubation
Control: corn silage with no additive; Cinn120: corn silage treated with 120 mg cinnamon essential oil per kg dry matter; Cinn240: corn silage treated with 240 mg cinnamon essential oil per kg dry matter; Thy120: corn silage treated with 120 mg thyme essential oil per kg dry matter; Thy240: corn silage treated with 240 mg thyme essential oil per kg dry matter; Mint120: corn silage treated with 120 mg mint essential oil per kg dry matter; Mint240: corn silage treated with 240 mg mint essential oil per kg dry matter; Oreg120: corn silage treated with 120 mg oregano essential oil per kg dry matter; Oreg240: corn silage treated with 240 mg oregano essential oil per kg dry matter; Cum120: corn silage treated with 120 mg cumin essential oil per kg dry matter and Cum240: corn silage treated with 240 mg cumin essential oil per kg dry matter.
IVDMD: in vitro dry matter disappearance after 24 h incubation.
PF: partitioning factor (mg DM disappeared per mL gas produced after 24 h incubation).
SEM: standard error of the means.
Moreover, pH of Mint240 favorably diminished. To our knowledge, no study has demonstrated the effect of mint essential oil on silage fermentation and its microbial population directly, although stimulatory effect of mint essential oil on growth of some strain of Lactobacillus bacteria was reported under in vitro condition (Voosogh et al. 2009). Results attained from the present study demonstrated that when silage treated with thyme essential oil, NH3-N concentration decreased indicating inhibition of deamination. Foskolos et al. (2010) reported a reduction in NH3-N concentration of ryegrass silage with high dose of thyme essential oil. Furthermore, McIntosh et al. (2003) illustrated that commercial blend of essential oil containing thymol (main active compound of thyme) reduced the rate of deamination under in vitro condition. Higher aerobic stability of treated silages confirmed findings of Chaves et al. (2012). In their experiment, silages treated with oregano or cinnamon leaf essential oils at 120 mg kg-1 DM remained stable for two weeks. Some PSMs such as carvacrol or thymol are able to inhibit the growth of some species of yeasts that are closely associated with aerobic spoilage (Knowles and Roller, 2001; Curtis et al. 1996). In addition to the direct effects of essential oils on silage fermentation, the current study also investigated the possibility of indirect manipulation of ruminal fermentation and methane production by using silages treated with essential oils. The chemical composition of silage can influence on the rumen microbial fermentation patterns and methane emissions (Navarro-Villa et al. 2013). Furthermore, some of essential oils have a good potential to alter rumen microbial fermentation and specially reducing rumen methanogenesis activity (Jahani-Azizabadi et al. 2011; Benchaar and Greathead, 2011) and ammonia producing bacteria in the rumen (McIntosh et al. 2003; Patra and Yu, 2014). Gas production of Oreg120 did not alter under in vitro condition that is similar to data which already reported by Chaves et al. (2012). However, it was decreased in Oreg240 after 24 h incubation. Some of previous studies investigating the effect of oregano essential oils on in vitro ruminal fermentation directly, revealed its wide-range spectrum of antimicrobial activity (Dorman and Deans, 2000; Walsh et al. 2003). Furthermore, effects of essential oils on rumen microbial populations are dose-dependent (Macheboeuf et al. 2008). Higher methane production per incubated DM in most of the treated silages was observed. These data were in contrast with findings of Chaves et al. (2012), who found no effect of some essential oils (when used as silage additives) on in vitro methane production. This inconsistency may be related to the type and dose of applied essential oils and type of substrate (Jahani-Azizabadi et al. 2014). Methane and total gas production had similar changes in each treatment and therefore, the ratio between them was not affected by experimental treatments. Increases in methane production in Cinn120, Oreg120, Cum120 and Cum240 were concomitant with decrease in IVDMD. Beauchemin and McGinn (2006) reported an adverse relationship between methane production and substrate degradation. However, lack of effects on substrate disappearance in response to some plant extracts that have affected methane production has also been reported (Sliwinski et al. 2002). In contrast, a reduction in IVDMD by application of cinnamon, cumin or oregano essential oils has also been reported (Jahani-Azizabadi et al. 2011). Oreg240 improved rumen fermentation as reflected by increase in PF. Higher PF in this treatment was consistent with its lower gas production and higher degradation of DM. Roy et al. (2014) also reported positive effect of higher dose levels of oregano essential oils on PF.
In conclusion, some of essential oils which used as silage additives had desirable effects on chemical composition of corn silage. Mint essential oils at 240 mg kg-1 DM had a protective influence on silage protein against deleterious deamination by decreasing pH of the silage. Furthermore, most of the applied additives improved aerobic stability of corn silage noticeably. Moreover, data obtained from the gas production expriment demonstrated the potential of oregano essential oil when used at 240 mg kg-1 DM to promote fermentation efficiency through reduction of gas production and increasing in vitro dry matter degradability.
The authors gratefully acknowledge the technical and financial support of Ferdowsi University of Mashhad.